A chiral molecule is a molecule whose mirror image is not superimposable on itself, in the same way that a right and left hand are not superimposable. Monitoring the progress of a chemical reaction, also called reaction monitoring, of chiral molecules involves the measurement of two quantities or properties of the reaction mixture. One quantity is the concentration (amount or fractional composition) of each of the molecular species present in the mixture. This quantity can be determined directly from the absorption spectrum across multiple wavelengths as a function of time during the course of the chemical reaction. The absorption spectrum will relate in some fashion to the concentration of the reacting chemicals. The second property is the percent enantiomeric excess (hereinafter % EE) of each of the chiral molecular species present. The % EE for one chiral molecular species is defined as the difference in concentration of one enantiomer (specific mirror-image form) of a chiral molecule minus that of the opposite enantiomer of the same molecule divided by the total concentration of the molecule (the sum of the concentrations of the two enantiomers) times 100%. The % EE of a single chiral molecular species can be measured using any form of optical activity such as optical rotation (OR) or circular dichroism (CD). When monitoring a chemical reaction of chiral molecular species, it may be desirable to monitor not only the concentration of a compound, but also the % EE of the enantiomers of the compound.
When creating chiral chemical compounds, particularly pharmaceuticals, it may be desirable to create more of a specific enantiomer of the compound and less of another. In such circumstances, it is advantageous to monitor the chemical reaction, so that the maximum of the desired product is produced. In practice, achieving this maximum may require careful management of the reaction conditions and variables, such as pressure, temperature, catalyst and chemical concentrations in the reaction chamber. A variation or change in any one condition could result in less of the desired product being produced.
When preparing to mass produce the compounds, great effort is devoted to determining the optimum conditions. Thus, it is advantageous to know the % EE when devising the conditions at which the production should take place. The method described herein allows real time monitoring of the % EE, allowing the conditions to be adjusted while observing the results of the adjustments. By extension, one will recognize that the % EE monitoring can be used during production to confirm that the proper product is being produced, and that the production is being maximized. If undesirable results are observed, the reaction conditions can be altered to achieve the optimum results. The monitoring methods of the prior art have been insufficient to allow real time monitoring of % EE.
There are two types of spectrometers typically used for measuring circular dichroism (CD), one in the visible and ultraviolet (Vis-UV) regions and another, called Fourier transform vibrational circular dichroism (FT-VCD) in the mid-infrared (MIR) region. OR instruments are also available commercially for fixed single-wavelength points in the Vis-UV region.
CD spectrometers in the Vis-UV regions of the spectrum (14,000 to 50,000 cm−1) are based on dispersive instrumentation that requires sequentially scanning the spectrum of interest through all the wavelengths to generate the desired spectrum. However, not all molecules of interest have the electronic transitions in the Vis-UV region as defined above. Additionally, time dependent studies are difficult because of the finite time needed to scan the spectrum, making dispersive techniques unsuitable for real time reaction monitoring.
Another method to monitor the course of a reaction is chiral chromatography. This involves the physical separation of the two enantiomers of the compound which requires time and the expense of maintaining the system including columns and eluting solvents over time. In addition it is not possible to monitor a reaction in real time using chiral chromatography.
CD spectrometers in the MIR region (800 cm−1 to 4,000 cm−1) of the spectrum are usually based on Fourier transform (FT) spectrometers in which all wavelengths are measured simultaneously. Infrared circular dichroism spectrometers measure the spectra of vibrational transitions of the chemical bonds in the molecules. These spectra, called vibrational circular dichroism (VCD) spectra, possess numerous transitions that are readily interpreted in terms of the structure and conformation of the molecule. Kinetic measurements of chemical reactions made with a FT-VCD spectrometer can be made simultaneously over the entire spectrum during one kinetic experiment, providing a distinct advantage over the dispersive scanning spectrometers, such as CD spectrometers in the Vis-UV region defined above. Thus FT-VCD is suitable for real time reaction monitoring.
The near-infrared (NIR) region of the spectrum lies between the MIR and the Vis-UV regions and spans the frequencies between 4,000 cm−1 and 14,000 cm−1. The NIR region is attractive because the underlying spectra are based on vibrational transitions and therefore possess the same sensitivity as the MIR region to molecular structure and conformation, as well as possessing a multiplicity of available transitions.
In the prior art, there are no available FT-VCD methods in either the MIR region or the NIR region to monitor the % EE of chiral species during a chemical reaction. Further, there is no indication in the prior art that FT-VCD measurements can be carried out in real time using a flow cell or any other method to monitor simultaneously for all chiral molecular species present the reaction.
The technology of dispersive scanning CD spectrometers is available in Vis-UV. However, it can only be used at one selected wavelength at a time, and thus lacks structural information about the molecule or molecules present. Additionally, if only a single wavelength is available to monitor the progress of a reaction involving two or more chiral molecules, such as with OR or CD in the Vis-UV region, it is not possible to follow simultaneously the % EE of any of the chiral molecules present if there is overlap of the OR or CD of these chiral molecules at that wavelength. The availability of simultaneously obtaining the infrared absorption (FT-IR) and VCD spectra using FT-VCD instrumentation permits the simultaneous determination of % EE of multiple species. The accompanying FT-IR spectrum as a function of time provides the simultaneous determination of the concentration of all chemical species present. When this information is combined with the FT-VCD measurements of the chemical reaction, the % EE of all chiral species present can be obtained by dividing the apparent % EE of each species by its concentration obtained from the ordinary FT-IR spectrum.
The method described herein involves analysis of the reacting species in real time using either a flow cell to bring the reaction mixture into the beam of the analytical instrument or fiber optics to bring the infrared light directly into the vicinity of the reaction vessel with or without the need to move some of the reaction mixture to a flow cell. The % EE of all the chiral species can be monitored simultaneously with the available vibrational FT-IR absorption spectra and FT-VCD spectra by normalizing the contributions of each species present to the VCD spectra by the concentrations of each species present as obtained from the FT-IR absorption spectra.
Early references to % EE determinations using VCD were based on measurements either using a dispersive scanning VCD spectrometer or a FT-VCD spectrometer. Most prior measurements were static, without the use of a flow cell, for a band of frequencies based on first recording the VCD spectrum of the pure enantiomer and then measuring the VCD of a sample that had undergone a chemical reaction or changes in % EE. IR spectra were used to determine the concentration of a single species or reaction product while the magnitude and sign of the VCD intensities could be used to determine the % EE of the enantiomers of the chiral sample molecule. Each of the measurements required a great amount of time to make and analyze. Because of the long scanning time required to measure the VCD spectrum in a dispersive spectrometer, even for one relatively narrow spectral region, there was no opportunity to follow the course of the reaction in time during the course of the reaction. For the FT-VCD measurements of % EE the use of a static cell prevented the possibility of monitoring changes in real time. The present invention allows both at-line and on-line real-time monitoring of continuously changing chemical reactions of chiral species through the use of a flow cell or fiber optic sampling.
Recent comparison of the relative merits of OR and VCD for the determination of % EE, indicate that OR was more accurate than VCD in many cases. However, cases in which either the OR is very small or more than one chiral species is present as a mixture, the % EE determined from the VCD measurement provides definite advantages with reasonable accuracy. In any case, OR is limited to single wavelength chiral monitoring in which it is not possible to follow the % EE of any chiral species, if two or more such species are present, with changing % EE.